Method and system for integrated mutliplexed modular photometry

ABSTRACT

Reusable network of spatially-multiplexed microfliuidic channels each including an inlet, an outlet, and a cuvette in-between. Individual channels may operationally share a main or common output channel defining the network output and optionally leading to a disposable storage volume. Alternatively, multiple channels are structured to individually lead to the storage volume. An individual cuvette is dimensioned to substantially prevent the formation of air-bubbles during the fluid sample flow through the cuvette and, therefore, to be fully filled and fully emptied. The overall channel network is configured to spatially lock the fluidic sample by pressing such sample with a second fluid against a closed to substantially immobilize it to prevent drifting due to the change in ambient conditions during the measurement. Thereafter, the fluidic sample is flushed through the now-opened valve with continually-applied pressure of the second fluid. System and method for photometric measurements of multiple fluid samples employing such network of channels.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of the pending U.S. patentapplication Ser. No. 16/216,397 filed on Dec. 11, 2018 and titled“Method and System for Integrated Multiplexed Photometry Module,” nowpublished as US 2019/0120748, which is a divisional of U.S. patentapplication Ser. No. 15/677,459, filed on Aug. 15, 2017 and nowpublished as US 2018/0088025. The disclosure of each of theabovementioned applications is incorporated by reference herein.

TECHNICAL FIELD

The present invention relates generally to systems and methods forconducting chemical, biochemical, and/or biological assays on a sampleand, more particularly, to multiplexed optical spectroscopy performed onsamples in microfluidic chambers.

BACKGROUND

Microfluidic devices and systems utilizing such devices employ smallcapillaries and/or microchannels and/or cuvettes associated or evenintegrated with a solid substrate to perform a variety of operations inanalytical chemical and biochemical applications on a very small scale.The small dimensionality of these systems facilitates sample processing(such as sample transport, analyte enrichment, reaction rate, etc.) thatuses less reagent volume and that takes up far less laboratory orindustrial space. Microfluidic systems thus offer the potential forattractive gains in efficiency of operation, and, consequently,substantial economic advantages.

A variety of spectroscopic techniques can be employed in conjunctionwith microfluidic devices, including those utilizing infrared (IR)radiation, visible light, and/or ultraviolet (UV) radiation, such aslight-scattering spectroscopy, for example. In research or industrialsettings, microfluidic devices are typically employed in biochemical orcell-based assays that use spectroscopic detection systems to quantifylabeled or unlabeled molecules of interest. Microfluidic devicesgenerally employ networks of integrated microscale channels andreservoirs (with the use of which fluid samples materials aretransported, mixed, separated and detected), and various optical systemsthat are embedded or externally arranged/coordinated with such networksfor optical recognition, detection, quantification, as well as othermanipulations of the fluidic samples.

There exists an unsatisfied need in such expansion of the assay menucapacity of a microfluidic photometric system that would manifest in thereduction of volume of a liquid sample (required for the photometricmeasurement) as well as improving the accuracy and precision of thephotometric measurement itself. Point of care integrated blood analysisinstruments and environmental monitoring instruments are but twoexamples of devices that would benefit from such expansion.

There also exists an unsatisfied need for a low per test cost (reusable,small volume) photometry system capable of performing a variety ofbiochemical assays (multiplexing) from a single sample at the point ofcare. The need of operable integration of such system with othercomplimentary analytical systems such as flow cytometry system tofurther simplify testing (by, for example, elimination of multipleinstruments/samples), capture economies of scale and scope (to reducethe overall cost) and enable decision making (for example, to obtaincomprehensive test data from a single sample) remains not addressed.

SUMMARY

Embodiments of the present invention provide a method for performing aphotometric measurement. The method includes the steps of (i)transmitting light from a first light source to a first photodetectorthrough a corresponding first cuvette containing a first fluid sampledelivered to the first cuvette from a corresponding first inlet; and(ii) transmitting light from a second light source to a secondphotodetector through a corresponding second cuvette containing a secondfluid sample delivered to the second cuvette from a corresponding secondinlet. The method also includes the step of acquiring data representingthe first and second fluid sample while at least one of the first andsecond fluid samples is prevented from being displaced, with respect toa respectively-corresponding cuvette, by (a) closing arespectively-corresponding valve in fluid contact with the at least oneof the first and second fluid samples on a first side of therespectively-corresponding cuvette, and (b) having the at least one ofthe first and second fluid samples under pressure on a second side ofthe respectively-corresponding cuvette, where such pressure is formed bya second fluid in contact with the at least one of the first and secondsamples. The closing of the valve may be effectuated while acorresponding fluid sample is under the above-specified pressure. Themethod further includes a step of removing the first and second fluidsamples from the first and second cuvettes throughrespectively-corresponding first and second outlets by openingrespectively-corresponding valves at the first and second outlets whilemaintaining the pressure. The first and second cuvettes are dimensionedto substantially prevent a formation of air-pockets therein while thefirst and second fluid samples flow therethrough. Alternatively or inaddition, the first and second cuvettes are dimensioned to minimizefluid-sample-to-fluid-sample carry-over due to said removing andsubsequent filling of any of the first and second cuvettes to notmaterially influence results of a subsequent step of acquisition of datarepresenting another sample measured in the same cuvette.

Embodiments of the invention also provide a related method forperforming a photometric measurement. The method includes temporarilystopping a flow of a first fluid sample through a first cuvette of afirst microfluidic channel of a microfluidic chip, for a first durationsufficient to carry out a first photometric measurement of an analyte inthe first fluid sample, to immobilize the first fluid sample and toprevent a first displacement of the first sample with respect to thefirst cuvette. Here, the microfluidic chip is structured to containmultiple substrates integrated with one another along theircorresponding surfaces to form an interface. The method further includescarrying the photometric measurement by:

(i) transmitting light from a first light source to a firstphotodetector through the first cuvette containing said first fluidsample that has been delivered to the first cuvette from a first inletthrough a first inlet channel that extends through the interface; and

(ii) while the first displacement is prevented, acquiring first datafrom the first photodetector, said first data representing said firstfluid sample. Furthermore, the method includes a step of completelyremoving the first fluid sample from the first cuvettes through a firstoutlet channel and a first fluidic valve operably cooperated with thefirst outlet channel.

Embodiments of the invention also provide a microfluidic device thatcontains first and second substrates integrated with one another alongsurfaces thereof to form a stack of substrates; a first microfluidicchannel including first inlet portion, first cuvette portion, and firstoutlet portion (here, at least one of said first inlet and outletportions traverses both of the first and second substrates); a firstfluidic valve in fluid communication fluidly connected to the outletportion; a fluidic well disposed upstream with respect to the firstcuvette portion in fluid communication with the first inlet portion.Here, the well has an internal volume and an aperture or orificeconnecting the internal volume with an ambient medium surrounding thewell. The well is equipped with a flap element dimensioned to reversiblyclose the aperture from inside the well when in a rest position, and toreversibly open said aperture in response to a force applied to the flapelement from the ambient medium inwardly to the internal volume.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be more fully understood by referring to thefollowing Detailed Description in conjunction with the Drawings, ofwhich:

FIGS. 1A and 1B are perspective and plan view illustrations of asimplified photometric microfluidic system employing a single cuvette.

FIG. 2 is an exploded perspective view of an embodiment of theintegrated photometric module of the invention.

FIGS. 3A and 3B are views of an embodiment, the configuration of whichfacilitates formation of air-bubbles inside a microfluidic cuvette.

FIGS. 4A and 4B are views of an embodiment which is configured tominimize and/or eliminate formation of air-bubbles in a microfluidiccuvette.

FIG. 5 is a diagram illustrating schematically an embodiment of amultiplexed fluidic network containing multiple cuvettes (each cuvettehaving corresponding inlets, high-resistance outlets) and alow-resistance main outlet channel.

FIGS. 6A and 6B are diagrams illustrating the fluidic model representingthe microfluidic network embodiment of FIG. 5.

FIG. 7 is a plot showing how the volume of the integrated photometrymodule (IPM) and the required volume V_(S) of the undiluted sampledepend from a thickness of a cuvette, for several values of dilutionratio D.

FIG. 8 provides empirically-obtained data evidencing the persistingsignal drift and associated uncertainty of acquired data thataccompanies measurements performed with microfluidic photometric systemsin which a fluidic sample is not spatially locked and immobilized withrespect to the housing volume containing such sample, as well ascomparison with data similarly-acquired with the use of an embodiment ofthe microfluidic photometric system configured to prevent a possibilityof fluidic sample movements during the measurement.

FIG. 9 schematically illustrates a concept according to which thefluidic circuitry of an embodiment of the invention is configured.

FIGS. 10A, 10B are schematic diagrams showing steps of preparing afluidic sample for photometric measurement, according to the idea of theinvention.

FIG. 11 is a flow-chart representing an embodiment of the method of theinvention.

FIG. 12 provides a schematic illustration of an embodiment of amultiplexed photometer system employing external valving.

FIGS. 13A and 13B schematically illustrate an embodiment of amultiplexed photometer system with internal valving, that has the only,single outlet in a fluidic manifold portion of the system. FIG. 13A:cross-sectional view of the system; FIG. 13B: top view of the fluidicmanifold portion of the system.

FIGS. 14A, 14B, and 14C provide schematics describing an embodiment of amultiplexed photometer system with internal valving, that has the only,single outlet in the photometer. FIG. 14A: cross-sectional view of thesystem; FIG. 14B: top view of the multiplex photometer module of thesystem; FIG. 14C: top view of the fluidic manifold portion of thesystem.

FIGS. 15A, 15B, 15C, 15D, 15E, 15F, 15G, and 15H provide additionaldiagrams illustrating the structure and principle of operation of anembodiment of the invention.

FIGS. 16A, 16B, and 16C contain plots representing results of empiricalvalidation of the operation of a system of an embodiment of theinvention and showing a comparison between the measurement resultsacquired with embodiment(s) of the invention and those provided byconventionally-used diagnostic equipment.

The sizes and relative scales of elements in Drawings may be set to bedifferent from actual size and scales to appropriately facilitatesimplicity, clarity, and understanding of the Drawings. For the samereason, not all elements present in one Drawing may necessarily be shownand/or labeled in another.

DETAILED DESCRIPTION

In accordance with an idea of the present invention, a microfluidiccuvette component of a network of microfluidic channels used in aphotometric module of the invention is structured such as to besubstantially completely filled and flushed, in operation, withoutleaving a volume of fluid that would substantially influence asubsequent measurement performed in the same fluidic network.Implementations of such cuvettes are many, including, for example,sample metering and/or conditioning. In this case, a cuvette (alsointerchangeably referred to as a chamber) is used to isolate arepeatable well defined volume of sample for further downstreamprocessing by, for example, appropriately incorporating fluidic valvesup-stream and down-stream with respect to the chambers to isolate thesample prior to processing. In another application, referred to hereinas “volume sensing”, an individual cuvette or chamber (that is adaptedto be filled and emptied substantially completely) is used to determinewhen a particular volume of fluid has been introduced into the system.Such volumes sensor could be placed at the outlet of the cuvette orchamber such that when the chamber is filled, the sensor is triggeredgenerating an indicator that the target volume has been reached. Used inany of such applications, an embodiment of the invention is configuredsuch as to ensure that the isolated is the cuvette volume of fluidicsample is spatially still/fixed/immobilized with respect to acorresponding channel/cuvette during the photometric measurement. Thissolution is provided, in part, by appropriately operating a fluidicvalves on one side of the cuvette to isolate the sample from the fluidicpressure downstream with respect to the cuvette. Alternatively, thesolution is provided by appropriately operating a fluidic valve on oneside of the cuvette while, at the same time, locking the fluidic sampleof interest in place with the use of pressure applied (with the use of adifferent fluid) to a front end and/or back end of the fluidic sample.

While the proposed cuvette element is operable and usable on its own, afluidic network of channels containing such fully fillable-and-emptiedcuvettes is also implemented. The network is adapted to operationallyisolate the individual cuvettes contained in different branches of thenetwork, is also implemented for use different applications including,for example, drug screening, facilitation of multi-reagent chemicalreactions, and photometric measurements. In the case of drug screeningfor example, the proposed fluidic network is adapted to differentiateamong individual cell cultures in a multiplexed cell culture sample. Anexample of the fluidic network employs an array of cell culture chambersthat can be individually stimulated with different chemicals but share acommon outlet. So designed network is configured to prevent cross-talkbetween the chambers, keeping each one in isolation. In anotherimplementation, the proposed fluidic network facilitates multi-reagentchemical reaction processes by isolating different components of achemical reaction from one another. When different branches of thefluidic network are flushed, the reaction would be initiated only in thecommon waste stream. In this manner, the order in which reagents areadded to the reaction solution are controlled, thereby facilitating thecontrol over the reaction products.

Related embodiments disclose examples of a microfluidic photometricapparatus configured, according to the idea of the invention, to takeadvantage, in operation, of an individual cuvette and/or of the proposedfluidic network. An implementation of the photometric apparatus has amultiplexed cuvette unit that is structured for repeatable andvolumetrically uniform fill-fix-in-space-measure-flush-and-re-useoperation substantially without forming air bubbles in the cuvettewhile, at the same time, providing sample aliquots with geometricalconstraints defined in such a fashion as to ensure that a pathlength oflight traversing the cuvette installed in the photometric apparatus issubstantially invariant.

References throughout this specification to “one embodiment,” “anembodiment,” “a related embodiment,” or similar language mean that aparticular feature, structure, or characteristic described in connectionwith the referred to “embodiment” is included in at least one embodimentof the present invention. Thus, appearances of the phrases “in oneembodiment,” “in an embodiment,” and similar language throughout thisspecification may, but do not necessarily, all refer to the sameembodiment. It is to be understood that no portion of disclosure, takenon its own and/or in reference to a figure, is intended to provide acomplete description of all features of the invention.

In addition, in drawings, with reference to which the followingdisclosure may describe features of the invention, like numbersrepresent the same or similar elements wherever possible. In thedrawings, the depicted structural elements are generally not to scale,and certain components are enlarged relative to the other components forpurposes of emphasis and understanding. It is to be understood that nosingle drawing is intended to support a complete description of allfeatures of the invention. In other words, a given drawing is generallydescriptive of only some, and not all, features of the invention. Agiven drawing and an associated portion of the disclosure containing adescription referencing such drawing do not, generally, contain allelements of a particular view or all features that can be presented isthis view in order to simplify the given drawing and the discussion, andto direct the discussion to particular elements that are featured inthis drawing.

A skilled artisan will recognize that the invention may possibly bepracticed without one or more of the specific features, elements,components, structures, details, or characteristics, or with the use ofother methods, components, materials, and so forth. Therefore, althougha particular detail of an embodiment of the invention may not benecessarily shown in each and every drawing describing such embodiment,the presence of this detail in the drawing may be implied unless thecontext of the description requires otherwise. In other instances, wellknown structures, details, materials, or operations may be not shown ina given drawing or described in detail to avoid obscuring aspects of anembodiment of the invention that are being discussed. Furthermore, thedescribed features, structures, or characteristics of the invention maybe combined in any suitable manner in one or more embodiments.

Moreover, if the schematic flow chart diagram is included, it isgenerally set forth as a logical flow-chart diagram. As such, thedepicted order and labeled steps of the logical flow are indicative ofone embodiment of the presented method. Other steps and methods may beconceived that are equivalent in function, logic, or effect to one ormore steps, or portions thereof, of the illustrated method.Additionally, the format and symbols employed are provided to explainthe logical steps of the method and are understood not to limit thescope of the method. Although various arrow types and line types may beemployed in the flow-chart diagrams, they are understood not to limitthe scope of the corresponding method. Indeed, some arrows or otherconnectors may be used to indicate only the logical flow of the method.For instance, an arrow may indicate a waiting or monitoring period ofunspecified duration between enumerated steps of the depicted method.Without loss of generality, the order in which processing steps orparticular methods occur may or may not strictly adhere to the order ofthe corresponding steps shown.

The invention as recited in the appended claims is intended to beassessed in light of the disclosure as a whole.

Photometric and radiometric methodologies (aggregately referred to, forthe purposes of this disclosure, using such terms as “photometry” and“photometric”, which includes fluorometric measurements such asperforming immunoassays using micro particles—ChemiluminescentMicroparticle Immunoassay or CMIA—as the antibody/analyte bindingsubstrate) have been widely adopted as tools for determiningconcentrations of analytes in both human and animal biological samplessuch as, for example, blood, urine, and saliva, to name just a few.(Photometric methods can also be used for environmental testing. Forinstance, groundwater can be tested for contamination due to variouschemical species.) In vitro diagnostic devices using photometricdetection techniques have been developed for a large variety of clinicalbiomarkers. In general, there are three classes of reaction schemes forclinical assays that are evaluated using photometric methods.

Chemical endpoint reactions involve the complete conversion of ananalyte using synthetic chemicals. The conversion results in a change inabsorbance of the sample, which is measured after the reaction hascompleted. The final absorbance of the sample is proportional to theanalyte concentration. Some analytes, the concentrations of which aredetermined with chemical endpoint assays, include hemoglobin, calcium,and total protein.

Enzymatic endpoint reactions also involve the complete conversion of ananalyte, such as glucose, for example. However in this case, theconversion is catalyzed by the presence of an enzyme. The absorbance ofthe sample is, again, measured after the reaction is completed and isproportional to the analyte concentration. Analytes the concentrationsof which are determined with enzymatic endpoint reactions includecreatinine, glucose, and bilirubin.

Enzymatic rate reactions involve the continuous conversion of an analytecatalyzed by an enzyme. Absorbance of a sample in this case is monitoredover time, and the rate of change of absorbance is proportional to theconcentration of an analyte. Enzymatic rate reactions normally requiretight temperature control to ensure that the reaction rate remainsconstant over the course of the measurement. Analytes the concentrationsof which are determined with enzymatic rate reactions include alkalinephosphatase (ALP), alanine aminotransferase (ALT), and chloride.

Based on Beer's law, according to which the absorption of light in asample is proportional to the concentration of the analyte, theabsorbance of light A_(X) ^(λ) at wavelength λ, caused by the presenceof species X at a concentration [X] along a path L through the sample,can be expressed as

$\begin{matrix}{A_{X}^{\lambda} = {{{ɛ_{X}^{\lambda}\lbrack X\rbrack}L} = {\log_{10}\lbrack \frac{I_{0}}{I} \rbrack}}} & {{Eq}.\mspace{14mu}(1)}\end{matrix}$where ε_(X) ^(λ) is the millimolar absorptivity of the species X at thedesignated wavelength. Accordingly, the concentration of thesought-after species can be expressed as

$\begin{matrix}{\lbrack X\rbrack = {{\log_{10}\lbrack \frac{I_{0}}{I} \rbrack}\text{/}( {ɛ_{X}^{\lambda}L} )}} & {{Eq}.\mspace{14mu}(2)}\end{matrix}$

The transmitted through the sample radiant power is determined byintegrating the light intensity transmitted by the sample over a rangeof wavelengths of interest and multiplying by the sensitivity of thedetector at those wavelengths. This can be accomplished in several ways.A broad spectrum light source may be used with a spectrophotometer as adetector which splits the transmitted light into component wavelengthsthat are individually detected and can be read at the wavelengths ofinterest. Alternatively, a narrow band wavelength light source may beused with a single point detector to absorb all of the transmittedlight.

Generally, the terms “sample”, “biological sample”, “chemical sample”and the like as used herein refer to a sample of fluid material that isassumed to contain an analyte of interest. For example, samples includevarious fluids such as various solutions, bodily fluids (such as wholeblood, serum, plasma, cerebrospinal fluid, urine, lymph fluids), andother fluids (such as, for example, cell culture suspensions, cellextracts, cell culture supernatants). A sample may be suspended ordissolved in, for example, buffers, extractants, solvents, and the like.Additional examples of samples are provided by fluids deliberatelycreated for the study of biological processes or discovery or screeningof drug candidates. The latter include, but are not limited to, aqueoussamples that have been doped with bacteria, viruses, DNA, polypeptides,natural or recombinant proteins, metal ions, or drug candidates andtheir mixtures.

Conventionally, to conduct optical spectroscopic and/or photometricanalysis, a sample should be placed in a cuvette that is used andreplaced after the measurement is complete. The currently employedmicrofluidic cuvettes possess shortcoming that substantially limit theirapplication in a multiplexed photometric system.

Indeed, conventional large scale systems use “open” cuvettes in whichsolution is directly pipetted into the cuvette (and not flown in througha permanently connected channel). These cuvettes are cleaned out aftereach use and reused with a cleaning solution via a robotic pipette.Alternatively, conventional point of care systems, which house thecuvette in a single use consumable, the cuvette is discarded at eachuse. The embodiments of the invention discussed below provide thesolutions employing a “flow in and through”, reusable cuvette), leadingto the advantage of lower consumable cost with respect to traditionalPOCT photometry systems. With respect to the open cuvette design oflarger conventional systems, the proposed below “flow through” systemeliminates the need to transport the sample to the cuvette roboticallyand requires a much smaller footprint.

In addition, in a multiplexed microfluidic photometric system adapted toperform parallel photometry measurements on multiple, generallydifferent analytes, a cuvette volume is an important figures of merit.The smaller the volume of a cuvette, the higher the degree of system andmeasurement multiplexing is possible for a given “footprint” of thedevice and the smaller the required sample volume. The term footprint,as used in this disclosure, would be readily understood—unless expresslydefined otherwise—as an area of normal projection of a given componentor element of the system onto a chosen plane. In order to makemeasurements of the sample reproducible, a cuvette must have a verywell-defined thickness or length (which translates to a well-definedsample path length through the cuvette). The path length of the cuvettedetermines the measurable concentration of an analyte for theinstrument. Accordingly, there is a need for a cuvette that isconfigured to ensure that the corresponding sample path lengthaccommodates the entire range of concentrations of interest.

In addition, a microfluidic cuvette must be configured such that, inoperation, it is completely filled with the sample at hand withoutintroducing air bubbles that obscure the optical path of light used forphotometric measurements. Air in the path of light leads to lightdiffraction, thereby causing errors in measurement of light absorbance.

Moreover, a cuvette is desired that lends itself to being re-used—incontradistinction with replaceable cuvettes of the related artdevices—and, therefore, “flushed” to sufficiently remove thejust-used/measured fluid sample to ensure that no substantialsample-carryover from one measurement to another. The latter requirementarises from a need to ensure that no sample-carryover contaminationoccurs from one measurement to the next.

The present invention stems from the realization that theabove-mentioned industrial needs are addressed with a microfluidicdevice configured to include a multiplicity of unidirectional-fluxcuvettes that share a common fluidic outlet, are devoid of valves, aredimensioned to substantially eliminate air-bubble formation in a flow offluid through each of the cuvettes, and that are subject to positivepressure facilitating substantially complete removal of the sampleresidue and, therefore, use and reuse of the same microfluidic chip.

FIGS. 1A and 1B provide perspective and plan view illustrations of asimplified photometric microfluidic system 100 employing a singlecuvette 110, providing a conveyor or container for a fluid sample (notshown) that is interrogated with light emanating from the light source120. Light from the source 120 passes through a spatial mask 130 havingan aperture 130A. In reference to FIG. 1A, light propagating between thesource of light 120 and the cuvette 110 along path 1 is transmitteddirectly through the aperture 130A on its way to a detector 140, whilelight following path 2 is shown to interact with (reflect off of) a sideof the cuvette 110. In one implementation, the source of light 120includes a 5 mm diameter LED, the opening 130A of the approximately 0.3mm thick mask 130 has a diameter of about 1.5 mm, a chamber of thecuvette 110 has a diameter of about 2 mm and thickness of about 1 mm,while the area of the detector 140 is about 1 mm².

As another preliminary matter, FIG. 2 provides an exploded perspectiveview of a simplified embodiment of a photometry module (IPM) accordingto an embodiment 200 of the invention that employs a multiplicity ofindividually addressable cuvettes 210 (at least some of which may besimilar to the cuvette 110 of FIGS. 1A, 1B). The cuvettes 210,configured for multiplexed photometry measurements, are disposed inassociation with a polymeric chip 212 and share a common waste outlet,while each of the individual cuvettes 210 includes a circularly-shapedchamber, as discussed in detail below. The IPM cuvette-chip 212 ishoused in association with an aluminum housing 220A, 220B that is usedas a heat sink to maintain a constant temperature of the system. Thesystem 222 (including the chip 212 and the sink elements 220A, 220B) isheated with Peltier heaters 224 located, as shown, around the exteriorof the aluminum heat sink housing 220A, 220B. The temperature of thesystem is monitored with a resistance temperature detector 226 (RTD)located within the bounds of the heat sink 220A, 220B in contact withthe polymeric chip 212. A feedback control loop (not shown) is employedto maintain the system at a constant temperature.

The heat sink 220A, 220B (with all the enclosure thereof) is disposedinside a plastic housing 230A, 230B configured to insulate the systemfrom the environment. A circuit board 240 containing an array ofphotodiodes 244 (such as, for example, an array of individualsingle-point photodiodes in T 1¾ packages) is mounted on one side of theplastic housing. In one implementation, the number N of photodiodesequals that of the cuvettes 210. The photodiodes may have, for example,square detectors with active areas of about 1 mm×1 mm and be protectedby flat optical windows. A complementary circuit board 250 containing acorresponding number N of narrow-band (or substantiallysingle-wavelength) LEDs 254 in T 1¾ packages with lensed tops is mountedon the other side of the plastic housing 230A.

As an option, a spatial mask (such as the mask 130 of FIGS. 1A, 1B, forexample) can be used to limit the area of light incident on thedetectors 244 from the light source 254 through the cuvettes 210. In oneembodiment, a substantially opaque at the wavelength(s) of interest masklayer can be sandwiched between the cuvette chip 112 and the aluminumheat sink portion 220A such as to have the apertures spatially alignedwith the individual cuvettes 210.

Optimization of a Single-Cuvette-and-Channel Geometry

It is appreciated that optimization of the operation of a microfluidicsystem depends, at least in part, on the ability of a user to utilize asample of a limited volume. To achieve such optimized operation, thevolume of the cuvette should not contain ‘dead space’ that is filledwith the substance of the sample but is not taking part in a photometricmeasurement. The required operational footprint of the cuvette, whichfacilitates elimination of such ‘dead space’, relates to the area of thephotodetector used for photometric measurements. In other words, thecuvette should be dimensioned such that every portion of light collectedby the photodetector has passed through the cuvette and that path lengthof such light through the cuvette is substantially the same for anyportion of the collected light. If this condition is not observed, thebackground noise associated with the measurement is increased and themeasurements system will have reduced sensitivity at the lower end ofthe sample-concentration range.

Another factor restricting configuration of a photometric system is apath length, for light propagating from a source of light through thesample being measured to a detector. Typical microfluidic photometricsystems are structured to ensure that such path length is on the orderof 1 cm. Some point-of-care blood analysis instruments, however, may beconfigured to utilize path lengths as small as a few hundred microns.

In further reference to FIG. 2, the entire operational volume V_(sys) ofa multiplexed photometric system is calculated asV _(SYS) =NAL+V _(C)  Eq. (3),where N is the number of cuvettes 210, A is the required area(footprint) of a single cuvette, L is the thickness of a single cuvette,and V_(C) is the volume of a network of channels adapted to providefeeding of the sample to the cuvettes 210 and removal of the waste fromthe cuvettes (and referred to as feeder-waste channel network, forsimplicity). The concentration of a diluted sample is given by[X]=[X]_(S)(1+D)  Eq. (4),Where the concentration of an undiluted sample is [X]s and the dilutionratio of the sample assay is defined as D. Based on Eqs. (1) and (4),A _(X) ^(λ)=ε_(X) ^(λ)[X]₁ L ₁=ε_(X) ^(λ)[X]₂ L ₂  Eq. (5A)[X]₂=[X]₁(L ₁ /L ₂)  Eq. (5B)and, in order to maintain a value of sample absorbance that remainsinvariant as the cuvette thickness changes, the dilution ratio D mustalso change as a function of the thickness of the cuvette:D ₂=(L ₂ /L ₁)(1+D ₁)−1  Eq. (6)

If the overall operational volume of the system is equal to the volumeof the diluted sample, the volume V_(S) of the undiluted samplecorresponding to the entire operational volume V_(sys) is determined asV _(SYS) =V _(S)(1+D)  Eq. (7)Assuming that a pathlength of light and the sample dilution ratio,corresponding to a chosen reference measurement method, are L_(R) andD_(R), respectively, the required volume V_(S) of the undiluted sampleis determined, from Eqs. (3), (5A, 5B), (6), and (7), to be reciprocalto the cuvette thickness L:V _(S) =L _(R)(NAL+V _(C))/(L+LD _(R))  Eq. (8)

Overall, the minimum operational value of the cuvette thickness isdetermined both by the necessity to measure the lowest concentrationanalyte (at the dilution ratio of the assay) and by the availability ofthe sample to be measure. As the cuvette thickness decreases, thenecessary sample dilution ratio for an assay decreases. If the dilutionratio of the sample is too low, there may not be enough volume of sampleto fill a multiplexed cuvette system.

Geometry of an entrance portion of the microfluidic network (forexample, a feeder channel that leads to the cuvette) and that of an exitportion of the network (a waste channel following the cuvette) areadditional factors defining the efficiency of operation of themicrofluidic system.

In reference to FIGS. 3A, 3B, 4A, and 4B, to ensure that no air bubblesare trapped and/or present in a cuvette filled with the substance of thesample and that the fluid flowing through the cuvette maintains acontinuous streamline along the wall, a surface defining a wall of thecuvette must be sufficiently smooth and define a tangent described by acontinuous function. In a specific embodiment, a wall of the cuvette isdefined by a surface that is differentiable (that is, has a derivative)at any point along the wall.

FIGS. 3A and 3B illustrate, not to scale, top and side views,respectively, of an embodiment 300 of a microfluidic network portionincluding a feeder channel 302, a cuvette 304 with a wall 306, and awaste channel 308. In order to reduce the ‘dead’ volume, the fluidicchannels that feed and empty the cuvette should be as small as possible.The cuvette 304 has an approximately rectangular profile defined by asubstantially step-like transitions between a chamber of the cuvette 304and the feeder/waste channels 302, 308 in a cross-sectional plane thatis parallel to both the main direction 310 of fluid sample flow and thedirection of propagation of light (shown as axis z in FIG. 3B). Inparticular, the transition angle A_(T) between the wall 306 and thebottom 312 of the cuvette 302 is substantially 90°. In contradistinctionto the embodiment of FIGS. 3A, 3B, FIGS. 4A and 4B illustrate anembodiment 400 the corresponding transitional angle A_(T) of which,defined by a non-zero-length T₁ transition between the feeder channel302 and the bottom 312 of the cuvette, is obtuse to define aramp-surface or wall 416, inclined with respect to the bottom surface312. Accordingly, the transition between the feeder or inlet channel 302and the cuvette 404 includes an entrance ramp region 406A. In oneimplementation, a minimum transition angle A_(T) of about 1440 between awall in transition region and the bottom of the cuvette ensures thatneither bubbles nor stagnant fluid are trapped in the corners and/or bythe edges of the cuvette 404 (the upper limit for this transition angleis, understandably, 180 degrees.) Similarly, the embodiment 400 can beadapted to contain an optional exit ramp region 406B, the transitionlength of which is denoted T₂ and which has a corresponding transitionangle (not shown in FIG. 4B)

It is appreciated that the smaller the microfluidic channels of thedevice, the more such channels are susceptible to clogging with thesubstance of the sample being measured. It is also appreciated thatshould an optimal channel size and/or dimension be chosen, suchsize/dimension will substantially minimize the total volume of thesystem.

In either of FIGS. 3A and 4A, the circular dashed line 350 identifiedthe area or footprint, of the corresponding cuvette 304, 404, that isrequired to ensure that all light leaving the source and reaching thedetector has passed through the cuvette (and thus the sample). Thedashed line 352 defines a parallelogram corresponding to tangents tofluidic cuvette walls.

According to an embodiment of the invention, the spatial rate ofwidening of the microfluidic network at the entrance of the cuvette, atthe transition region between the inlet portion and the cuvette, issufficiently low to ensure that the fluid sample proximate to the wallof the cuvette doesn't separate from the wall and form bubbles near theedges of the cuvette. For example, the fluid sample may be controlledusing a boundary layer or surface tension effects. This spatial rate ofwidening of the transition portion is defined, for example, by an angleof widening A_(W) formed by a wall in the transition region with respectto an axis of at least one of the inlet and outlet portions; the valueof A_(W) is smaller than a threshold angle value Θ. If, as illustratedin FIG. 3A, such angle A_(W) between the wall 306 and the direction ofthe flow 310 exceeds the operationally corresponding threshold value Θ,the nature of interaction between the fluid stream in the separationzone 316 and the wall 306 is likely to promote formation of the bubbles320 (region B in FIG. 3A) in the cuvette 304B. In contradistinction withthe embodiment of FIGS. 3A and 3B, the embodiment of FIGS. 4A and 4B,having a cuvette 404 that is characterized by A_(W)<Θ, does not promoteformation of the bubbles. In one embodiment, the value of the thresholdangle Θ is about 36 degrees and, therefore, 0≤A_(W)≤36°. In anotherembodiment, the value of the threshold angle is about 30 degrees and0≤A_(W)≤30°, and in an alternative embodiment the value of the thresholdangle is about 25 degrees and 0≤A_(W)≤25°. In a specific case whenA_(W)˜0° and the depth of the feeder channel 302 approximately equals tothe depth of the cuvette 304, 404 (not shown), there is substantially nopotential for trapping air bubbles or leaving stagnant fluid at thebottom of the cuvette.

Furthermore, an embodiment of the IPM of the invention (such as the IPM200 of FIG. 2, for example) is optionally equipped with a unit providinga positive air flow through the microfluidic network including thecuvettes 210, and each of the cuvettes 210 is adapted to ensure thatthere are no ‘stagnant’ areas in the cuvette in which the residue of thefluid sample remain after the cuvette has been flushed with air.Accordingly, and in further reference to FIGS. 4A, 4B, the radius 408 ofcurvature defined by a cuvette wall 410 in a cross-sectional plane thatcontains the axis 310 of fluid flow and that is substantially normal tothe optical axis (locally defined as z-axis) should be maximized. In oneexample, where the footprint of the cuvette of about 2 mm (as defined bythe dashed circle 350) implies a maximum radius of curvature of about 1mm for the channel walls in the middle of the cuvette region(approximately at a mid-point between the inlet 302 and outlet 308 ofthe cuvette).

Optimization of a Multiple-Cuvette-and-Channel Multiplexed Geometry

Embodiments of the invention employ reusable microfluidic chips orelements that combine, in a spatially multiplexed fashion, multipleindividual cuvettes each of which is adapted for a designated uniquetype of measurement. For example, multiple individual cuvettes on thesame chip may be used for contemporaneous measurements of the same typeof sample the concentration of which is different in different cuvettes.In a related example, multiple individual cuvettes on the same chip maybe used for contemporaneous measurements of samples of different typesor nature (for example, samples containing different analytes). Ineither case, to use the smallest possible volume of a sample in anindividual cuvette, the ‘dead’ volume of such cuvette is minimized, asmentioned above. A person of skill in the art will appreciate that therequired operational independence of the individual butstructurally-multiplexed cuvettes from one another begs a question ofhow to preclude different sample aliquots in different individualcuvettes from mixing with one another and, by virtue of such mixing,introducing an error in the measurements. In addition to one fluidmixing with another, one should also appreciate the need to overcomefilling and cleaning of individual cuvettes independently due to thevarying time constraints of each assay (reaction/incubation times) withrespect to sample processing logistics in a multiplexed system.

This requirement becomes even more stringent if another requirement isimposed to not remove the reusable microfluidic chip from thephotometric apparatus between immediately sequential measurements.

Put differently, the complexity of these problems can be phrased asachieving the operational multiplexing of cleanable cuvettes on the same(optionally non-removable from the photometric apparatus) chip, while(i) minimizing the number of necessary fluidic connections on the chip,to reduce the overall footprint of the chip and the ‘dead’ volume of thecuvettes and (ii) ensuring that samples in individual cuvettes aresubstantially isolated from one another. According to an embodiment ofthe invention, a solution to this complex of problems is provided bymerging the individual outlet channels of individual cuvettes into acommon outlet for the overall multiplexed system of cuvettes. Thefollowing discussion is provided in reference to FIG. 5 providing adiagram of an embodiment 500 of a multiplexed microfluidic chip for usewith an embodiment of the photometric apparatus of the invention.

Sample Isolation. The embodiment 500 shows an example of multiplexing ofindividual microfluidic elements each of which includes a correspondinginput channel or inlet 502(a, b, c, d, e, f, g, h, i, j), a cuvette504(a, b, c, d, e, f, g, h, i, j), and a corresponding individual outputor outlet 508(a, b, c, d, e, f, g, h, i, j). For simplicity ofillustration, only some of the above-mentioned elements are labeled inFIG. 5. The individual inlets 502 a through 502 j serve a purpose ofoperational isolation of individual cuvettes 504 a through 504 j so thatdifferent samples in these cuvettes do not contaminate one another. Forsimplicity of the fluidic manifold that will distribute and returnsamples, the individual outlets 508 a through 508 j are merged to andshare a common chip outlet 516. To operationally isolate the samples inthe cuvettes 504 a through 504 j, the fluidic resistance of theindividual cuvette outlets 508 a through 508 j must be sufficient toensure that pressurized fluid in the main outlet channel 516 flows outof the device (along the arrow 518) through the common outlet ratherthan back, up-the-stream through another cuvette (for example, throughthe outlet 508 a towards the cuvette 504 a). The fluidic path lengthcorresponding to an individual outlet channel 508 a through 508 j mustbe sufficiently long to prevent diffusive mixing of the samples inindividual cuvettes 504 a through 504 j.

In reference to FIGS. 6A and 6B, showing examples of two schemesdescribing the fluidic model of the embodiment 500 of FIG. 5, thefluidic resistance R1 of the individual outlets 508 a through 508 j is,in one implementation, approximately 2 to 3 orders of magnitude higherthan the resistance values R2 of segments 524 ab, 524 cd, 524 ef, 524gh, and 524 ij, of the main outlet channel 516, that are located betweenthe cuvette pairs (504 a, 504 b), (504 c, 504 d, (504 e, 504 f), (504 g,504 h), and (504 i, 504 j), respectively. In one implementation, theratio of the resistance values R1/R2 is about a number of the individualcuvettes in the embodiment or higher. In different implementations, theratio of resistance values R1 and R2 may range between about 200 andabout 10,000. Furthermore, the entire measurement system, which includesthe embodiment 500, is preferably a closed system with no air in anyline in order to prevent residual flows. If there is any air in thesystem, it will compress during pressurization of the system and thenwhen the system is de-pressurized, the air will expand causing residualflows which can lead to sample contamination. The nodes Ni within thesystem of FIG. 6B represent the locations at which the fluidic pressureis approximately the same.

Examples

In one implementation, and in further reference to FIGS. 2, 4A, 4B, and5, an individual cuvette in a multiplexed chip 212, 500 used in the IPM200 has a footprint defined by a circle with a diameter of about 2 mm.FIG. 7 is a plot showing a volume of the required undiluted sample as afunction of the cuvette thickness and the reference dilution ratio,according to Eq. (8). According to FIG. 7, a cuvette with thickness ofabout 1 mm optimizes the reduction of the ‘dead’ volume of the overallmicrofluidic component system while maintaining, at the same time, anundiluted sample volume below about 0.02 mL. Therefore, FIG. 7 providesan illustration to an embodiment in which the operation of themultiplexed microfluidic chip 212, 500 is optimized for the cuvetteshaving thickness of about 1 mm. In the example of FIG. 7, the entrancechannels (302, 502 a through 502 j of the cuvettes (404, 504 a through504 j) each have a depth of about 1 mm and a width of about 0.5 mm. Thetransition angle A_(T) corresponding to the individual inlet transitionarea 406A is chosen to be about 180°, as discussed in reference to FIG.4B. Each of the individual inlets (302, 502 a through 502 j) expandsinto the corresponding cuvette (404, 504 a through 504 j) at an angleA_(W) of about 32°, as discussed in reference to FIG. 4A. The walls ofan individual cuvette have a curvature radius 408 of about 1 mm. Theindividual exit channels (308, 508 a through 508 j) have a width ofabout 0.25 mm, a depth of about 0.05 mm, and a length of about 26 mm.The transition region 406B from an individual cuvette area to thecorresponding exit channels (308, 508 a through 508 j) has a length T₂of about 1.9 mm. The transition angle A_(T) corresponding to the outlettransition region 406B is chosen to be about 153°. The main outletchannel 516 has a depth of about 0.25 mm and a width of about 0.5 mm.The distance between pairs of individual outlet channels along thelength of the main outlet channel is about 5.4 mm.

In one implementation, and referring again to FIG. 2, the IPM 200 isoperated as follows. The entire system is heated to approximately 37° C.(for example, for about 5 minutes) until a steady state is reached. Oncethe temperature of the system is stabilized, a dark reference voltage ismeasured for each detector 244 by determining the voltage correspondingto each detector when the respectively corresponding LED 254 is turnedoff. The IPM 200 is initially empty (contains air) prior to anymeasurement. Blank solutions are pre-loaded into a sample manifold (notshown in FIG. 2). The manifold is connected to the plastic cuvette with3″-6″ long rigid tubing (such as, for example, the one by PEEK, 1/32″OD, 0.015″ ID) followed by 3″-6″ long flexible tubing (such as that ofTygon, 0.06″ OD, 0.02″ ID). The manifold is pressurized to about 1 barand the blank solutions flow into their respective cuvettes. The flow isstopped by venting the manifold to atmospheric pressure when liquidbegins to flow out of the outlet tube (in one example, in about 5 to 15seconds). The system is then closed by clamping the flexible tubing toensure that there are no air pockets in the system (such as the headspace in the manifold).

The transmission of light through the blank solution in a single cuvetteis measured by turning on the LED 254, corresponding for that cuvette,waiting for a short period of time (such as 5 ms, for example),recording the voltage of the photodetector 244, turning off the LED 254,and waiting for another period of time (for example, 200 ms). Thisprocess is repeated three times and the average detector voltage forthat cuvette is determined. The dark reference voltage for that detectoris subtracted from the average voltage and the result is recorded. Theprocess is repeated for each of the ten cuvettes in series.

Once the blank solutions have been measured, the sample vials areremoved from the manifold and tubing is unclamped. The manifold is againpressurized to 1 bar and the system is flushed with air until all of theliquid is removed (for about 5 to 15 seconds). The samples are thenloaded into the manifold in the same manner as the blank solutions.

The transmission of light through the samples is measured in the samemanner as for the blank solutions. The absorbance of the samples isdetermined by calculating the logarithm of the ratio of thetransmittance value corresponding to the blank solution to thetransmittance value of the sample (corrected with the dark referencevalue, as already described).

For endpoint reactions, three measurements are taken for each sample andthe averaged absorbance value is used to calculate the sampleconcentration. For rate reactions, the absorbance of the sample iscontinuously monitored (the system cycles through all of the cuvettesuntil stopped) and the rate of change of absorbance is used to determinethe sample concentration.

The IPM embodiment 200 of FIG. 2, structured as described above inreference of FIG. 7, requires at least 0.02 mL of the fluid sample (forexample, blood, serum, saliva diluted with reagent and/or buffer) tooperate. In order to make accurate measurements of sample absorbance,the liquid sample in any given cuvette must not be contaminated by othersamples. This means there can be no residual flows in the system. Thetube clamping described in the previous section takes care of thisrequirement.

For enzymatic rate reactions, the sample and reagent are mixed outsidethe system at a temperature significantly lower than the chosesteady-state temperature of operation (which, in the provided example,is about 37 C) in order to suppress the enzymatic rate reaction. Whenthe sample is flown into the cuvette, it is warmed to 37 C as quickly aspossible in order to make an accurate constant rate measurement. Assaychemistries for this system should preferably be adapted to becompatible with that process workflow.

In order to make accurate sample measurements, the LEDS 254 andcorresponding electronic circuitry are configured to ensure that theoutput LED intensity does not drift over time. In particular, the dutycycles (on time/cycle time) of the operation of the LEDs 254 are chosento be low enough to ensure that the LED intensity doesn't drift. In oneexample, the reported operation of ‘5 ms on/200 ms off’ satisfies thisrequirement for all of the LEDs currently used in the IPM system 200.

As was already alluded to above, one of the problems persistentlyaccompanying the operation of a microfluidic photometry module duringthe measurement is a drift (or spatial shift, or repositioning) of afluidic sample housed in a volume through which light, used formeasurement, is passed between the source of light and the opticaldetector (such as a cuvette portion of the microfluidic channel). Whileone might expect that, under some circumstances (and depending on assaychemistry), capillary forces would help maintaining the volume of thefluidic sample firmly in place, the fact that at least one of the frontand back ends or interfaces of the fluidic sample in the channel isconventionally left in its natural state (or free, or unattended) allowsfor minute movements of the sample caused by any disturbance occurringin the ambient media surrounding the sample during the measurement.Alternatively or in addition, minute movements of the sample filling thecuvette may also be caused by differences in pressure levels on oppositesides of the sample (up- and downstream with respect to the cuvette).Such movements or drift occurs at amplitudes practically sufficient toperturb the measurement and cause such uncertainty of the results thatoften shed a doubt on accuracy and/or repeatability of the measurement.

It would be appreciated from further disclosure, that implementation ofthe embodiments of the invention increases the quality and reliabilityof photometric, fluorometric (for example, chemiluminescent), andturbidimetric analyses of sample to determine concentration ofanalyte(s) (in a non-limiting example—for in vitro diagnostic todetermine concentrations of specific analytes in a blood sample).Specifically, embodiments of the invention advantageously improve thequality of multiple photometric and/or turbidimetric and/or fluorometric(for example, chemiluminescent) measurements that have to be performedin the same cuvette of the same chosen channel of the channel network,requiring the system to have both inlet and outlet portions of thechannel so that multiple samples can be allowed to flow through. Twoexamples of such a requirement are: 1) a situation when the cuvette isreused for testing of multiple samples; and 2) when a calibrant ismeasured in the same cuvette prior to measuring the sample of interest.It has been observed, in practice, that flow through the cuvette(s) issusceptible to residual flow created by differences in pressure upstreamand/or downstream of the cuvette and/or capillary forces, and that thisresidual flow detrimentally affects the analysis of the sample. Theobserved effect is of particular importance in the case of assays thathave to be maintained at a specific temperature (and/or within aspecific temperature range) within the cuvette—such as is the case, forexample, with enzyme catalyzed reactions. Furthermore, in case whenmultiplexed configurations of microfluidic channels are employed thatconnect, downstream, to a common waste collection reservoir (storagevolume), it may be required to temporarily isolate one portion of theoverall system from another to prevent the fluidic pressure in one ofthe channels from influencing the fluid flow in other channels. Notably,in some cases, in the same network of channels, it may be important toensure that any combination of analyses (photometry, turbidity,chemiluminescence) can be performed simultaneously.

Empirically-procured evidence of such seemingly-insignificant problem isprovided by FIG. 8, where the photometrically-acquired optical datarepresenting absorbance of the sample (“absorbance units”) are plottedas a function of time. The plots presented in this Figure illustrate twosituations: the one often occurring during the conventionally-configuredphotometric measurement (that is, without a precaution of “locking” thesample in place), and the other corresponding to the implementationcarried out according to the idea of the invention (that is, with thefluidic sample “locked” in its position to avoid the drift caused byexternal disturbances and/r changes of environment, as will be discussedin detail below). The first situation is represented by the group 810experimental curves, each of which exhibits a sharp transition such astransition 820A of the curve 820, during which the fluidic sample beingmeasured experiences a minute spatial drift disturbing and changing thereading at the optical detector. The second situation is represented bythe group 830 of the curves, each of which exhibits a clearly monotonic,differentiable behavior. As will be readily appreciated by a skilledartisan from the discussion below, the drastic change in reliability ofthe measurement is caused by the implementation of the idea of theinvention.

Considering the miniscule amounts/volumes of fluidic samples that thedescribed above embodiments of the invention are capable of measuring,such drifts or shifts (see transition 820A of curve 820) present aproblem that begs a reliable solution. According to the idea of theinvention, the fluidic sample—once positioned in a cuvette in a fashionappropriate for photometric measurement—is spatially locked or fixed orimmobilized in its position by intentionally preventing a possibility ofeither of its front or back interfaces to move. This is achieved byusing a fluidic valve at an identified point on one side of the sampleto prevent the sample from moving pass such identified point and toisolate the downstream compressive fluid (gas, liquid) from the sampleduring the measurement. In a related embodiment, this achieved by usinga fluidic valve at an identified point on one side of the sample toprevent the sample from moving past such identified point while, at thesame time, applying a fluidic pressure to the other, remainingend/interface of the sample. The latter can be carried out with, forexample, a flow of gas passed through a given channel of the network ofthe microfluidic channels in the direction of propagation of thefluid-sample-being-measured through the system. In a related embodiment,however, the system may be appropriately configured to employ a flow ofliquid.

A person of skill in the art will readily appreciate that the componentsand/or elements of the system and the overall system itself generallycan be and are intended to be implemented not necessarily in a single,common for all components/elements structural layer of the system. Inone example, multiple microfluidic channels of the overall network ofchannels may be disposed in a single substrate (and even in a singleplane of such substrate), such as in the case already discussed inreference to FIG. 5, for example. In a related case, however, asdiscussed below, the operability and efficiency—as well as cost—of theoverall system are increased by purposefully and intentionally extendingdifferent elements of the system (such as different portions of themicrofluidic network) into and/or through multiple substrates and/orplanes—often transverse to one another and generally made of differentmaterials—in a fashion that allows for substantial minimization of afootprint of a given microfluidic chip.

Outlets of individual, constituent channels of the network ofmicrofluidic channels of the system (such as an outlet portion of thechannel, through which the already-measured fluidic sample is propagatedfrom the cuvette) may be structured to lead to a main outlet channel(contained within the same chip carrying the network of channels andshared by multiple individual constituent channels, by analogy with thestructure illustrated in FIGS. 5, 6A). The system may be furtherconfigured such as to direct the main outlet channel to a storage volumecollecting used/measured fluidic samples. Alternatively or in addition,such storage volume can be formed in direct contact (be permanentlyintegrated) with the chip containing the network of channels (forexample, be formed on a substrate already carrying at least a portion ofthe network of channels). In a related embodiment, however, the storagevolume—if present—is formed on an independent piece of material that isnot inseparably integrated with a portion of the microfluidic networkbut, to the contrary, is disposed on a hardware component that can beremoved from the system on its own (to be disposed of, for example).

Putting aside, for a moment, the specific description of how thecomponents of the network of microfluidic channels are oriented withrespect to one another, the concept of locking the fluidic sample inplace to avoid a drift of the sample during the measurement can beillustrated with the schematic of FIG. 9. Here, a plurality ofconstituent channels 910, 920, 930 are shown, each having acorresponding inlet portion 910A, 920A, 930A and a corresponding outletportion 910B, 920B, 930B, with a corresponding cuvette portion 910C,920C, 930C in between. In this example, the cuvette portions 910C, 920C,930C are shown to be formed on a dedicated substrate 940 (which may ormay not be the same substrate that carries at least one of the inlet andoutlet portions, too). The outlet portions 910B, 920B, 930B are shown tolead to an area 950 (which, depending on the specific implementation maybe a main outlet channel leading, in turn, to the storage volume, or astorage volume itself). Arrows in FIG. 9 indicate a direction of flow offluidic sample(s) through corresponding channel(s) during the operationof the photometric system of the invention. As schematically shown,fluidic valves 960, 970, 980 are used on one side of channel(s)—shown,in FIG. 8, downstream from the cuvettes 910C, 920C, 930C—to reversiblyblock path(s) for sample(s) moving along the channel(s). Once themovement of a given fluidic sample down a given channel is stopped byclosing a corresponding valve, the locking of the sample in place isfurther effectuated by pressuring the channel at the other side withrespect to the corresponding cuvette.

The principle of preparation of a given microfluidic channel for aphotometric measurement of the fluidic sample contained therein,arranged according to an embodiment of the invention, is furtherillustrated in reference to FIGS. 10A and 10B. Here, for simplicity ofillustration, only one of the channels of FIG. 9 is considered and shownin side view. With the downstream valve 960 open, fluid (for example,gas) pressure 1010 is applied to the back, tailing interface of thefluidic sample 1020 in the inlet 910A to force the sample 1020 throughthe cuvette 910C and the valve 960. The fluidic pressure can be formedwith the use of, for example, a pump that is appropriately connected tothe inlet 910A upstream with respect to the cuvette 910C. Prior totaking the analytical measurement, the photometric system (some ofprincipal components of which are shown here as a light source 1030, andoptical aperture 1040, and an optical detector 1050) is used to detectthe progress of the front interface of the fluidic sample 1020 towardsthe valve 960. Referring no to FIG. 10B, once the front end 1020A of thefluidic sample 1020, pushed from the back with the pressure front 1010,passes through the valve 960, the valve is shut to prevent any furtherforward drift/repositioning of the sample 1020 while, at the sampletime, the fluidic pressure 1010 applied to the back end 1020B of thesample 1020 is maintained to prevent the repositioning of the end/tailinterface of the sample 1020 on the other side of the cuvette 910C. Thevolume of a sample contained, under such conditions, between theinterfaces 1020A, 1020B defines the sample volume used for a photometricmeasurement; it is preferred to dimension the channel and/or valveand/or pressure applied to the tailing interface of the sample in such afashion as to minimize this volume. One guideline for minimization ofthe sample volume may be to minimize the distance between the valve andthe cuvette portion of the channel.

Referring again to FIG. 10B, several modalities can be used forreal-time determination of the position of the fluid front interface1020A of the sample 1020 downstream of the valve 960, according to anembodiment of the invention. For example, the front 1020A can belocalized with the use of optical detection (based on absorption,refraction, or scatter of light, directed to a point downstream of thevalve and the use of an external optical detection unit). Alternativelyor in addition, the propagation and/or location of the front 1020A passthe valve 960 can be determined with the use of electrical detection bymeasuring the impedance value, for example. Alternatively or inaddition, the mechanical methods can be used as well such as thedetermination of pressure with the transducer-containing module inoperable communication with a portion of the microfluidic channel and/oremploy of a limit-switch, as may be known in the art. The use ofpredetermined parameters of process variable (such as fluidic pressure1010 and time) is further made to drive the sample 1020 beyond the valveafter the feedback signal is detected by the appropriate sub-system ofthe photometric system.

FIG. 11 contains a schematic chart representing some of the steps of amethod of the invention. A person of skill will appreciate that anembodiment of the method contains at least some of the followingprocessing elements. At step 1110, fluidic pressure is applied to thetailing end of the fluidic sample in a channel to move the sampletowards the cuvette. At 1120 (and in further reference to FIG. 10A), theoptical system of the photometric module is used to observe the fillingof the cuvette with the fluidic sample and/or to confirm that thatcuvette has been filled without the formation of air-bubble within thecuvette. The continually-applied to the tail interface of the samplepressure is then used to move a portion of the spatially-continuoussample pass the cuvette through the downstream valve, 1130. Once thefront interface of the sample is observed to have moved pass the valve,the valve is closed at 1140 to ensure that the sample can no longer movein the forward direction while, at the same time, the tailing interfaceof the sample is maintained under the fluidic pressure directeddownstream to prevent the sample from moving backward and to avoid aspatial drift or repositioning of the sample due to a change of ambientconditions. (In a related implementation, however, it may be possible toremove the upstream pressure of the system once the downstream valve isclosed, while preventing the backflow of the fluid. It may be preferredto retain the stored energy of the upstream pressure for efficiency ofoperation of the overall system).

The so-fixed-in-place fluidic sample is then subjected to thephotometric measurement at 1150 to determine sought-after parameter(s)as discussed above. Upon the conclusion of the analytical measurement,the valve is opened at 1160 to allow for the forward movement of thesample out of the cuvette and pass the valve under the fluidic pressureapplied to the tailing end of the sample. During the ridding 1170 of theportion of the channel (e.g., cuvette) of the fluidic sample, theparameters of the fluidic pressure applied to the tailing end of thesample may be judiciously modified to ensure that such channel portionis completely emptied.

The process of handling a fluidic sample upon its propagation throughthe network of microfluidic channels and photometric measurement ofanalyte(s) contained in the sample can be effectuated with the use ofseveral embodiments of the microfluidic chip carrying such network. Thevalve(s) of the embodiments may be generally arranged as externalvalve(s) (that is, arranged externally to the overall, whole network ofchannels such as to control the fluid flow through a main outlet channelthat is common to or shared by individual outlet portions of constituentchannels of the network). Alternatively, the valve(s) can be arranged asinternal valve(s) (that is, valve(s) governing the fluidic flow throughoutlet portions of individual constituent channels of the network.Further below, three related non-limiting examples of so configuredembodiments are discussed in reference to FIGS. 12; 13A and 13B; and14A, 14B, 14C, which present implementations in which the channels ofthe network are configured to penetrate multiple structural levels andare not necessarily belong to a single carrying level or substrate.

FIG. 12 schematically illustrates, in a side cutaway view, an embodiment1200 of the microfluidic chip in which an individual channel 1210 (thatincludes the inlet portion 1210A, the cuvette portion 1210B, and theoutlet portion 1210C) is structured to pass through at least threestructural layers 1220, 1230, and 1240, operationally integrated one ontop of another for proper photometric measurement. (The side view of theembodiment 1200 shows two channels—1210 configured on the right-handside of the chip as shown and the other channel disposed substantiallysymmetrically with respect to the channel 1210). In particular, thecuvette portion 1210B is formed in the upper (as illustrated) layer1220, defining a fluidic photometry module through which light istransmitted during the photometric measurement and that is carried on amounting surface of the substrate 1230. The inlet portion 1210A leadingto the cuvette 1210B, on the other hand, is directed to penetratethrough the fluidic manifold layer 1240 (in directions parallel and/ortransverse to a mounting surface of the layer 1240), the substrate 1230connected to the mounting surfaces of the layers 1220, 1230, and atleast in part through the layer 1220 where it merges to the cuvette1210B. Similarly, the outlet portion 1210C of the channel 1210 emergesfrom the cuvette 1210B to penetrate through the photometry module layer1220, the mounting substrate 1230, and the fluidic manifold layer 1240on its way to the main outlet channel (main outlet) 1250 that, in thisembodiment, is common to and shared by multiple constituent fluidicchannels. Before merging to the main outlet 1250, the outlet portion1210C is fluidically engaged with the external valve 1260, which isconfigured to block the fluid flow through the outlet portion 1210C whenclosed, as discussed above. (A Clippard valve can be used for thispurpose in one case, as illustrated.) During the operable integration ofthe layers 1220, 1230, 1240, surface sealing layers such as gasket(s)and/or O-rings can be installed between the facing-each-other surfacesof these layers to provide for appropriate fluidic seals.

FIGS. 13A and 13B provide schematics of a related embodiment 1300. Asshown, a constituent channel 1310 includes an inlet portion 1310A, acuvette portion 1310B, and an outlet portion 1310C, each of theseportions of the channel 1310 formed in at least one of the structurallayers 1320, 1330, and 1340 that are (optionally, reversibly) integratedwith one another through the sealing layers 1344A, 1344B (such asgaskets, for example). Here, each constituent channel is complementedwith a respectively-corresponding valve (as shown, the channel 1310 iscooperated with the valve 1360), which is configured in the fluidicphotometry module or layer 1320. FIG. 13B schematically illustrates atopview of the fluidic manifold structural layer 1340 of the chip 1300,with the main, shared by the constituent channels outlet 1350(optionally leading to a fluid storage volume, not shown).

FIGS. 14A, 14B, and 14C illustrate an embodiment 1400 structured byanalogy with those of FIGS. 12, 13A, but exhibiting a single, shared byindividual outlet portions of the constituent channels, main outlet(outlet port) 1450 that is located at least in part in the photometrymodule layer 1420. Here, an individual channel 1410 includes, as before,the inlet portion 1410A disposed across the structural layers 1420, 1430(which is a mounting substrate), and 1440 (which is a fluidic manifold).In a fashion similar to those of the embodiments 1200, 1300, thestructural layers 1420 and 1440 are shown mounted on the substrate 1430with preferably interdisposed surface sealing layers 1444A, 1444B thatensure lack of leakage of the fluidic sample(s) flowing throughconstituent channels of the network of the chip 1400. The individualchannel 1410 also include the cuvette 1410B (carried in the photometrymodule layer 1420), and the outlet portion 1410C passing through atleast the layer 1420. The outlet portion of each of the individualchannel of the network of channels is operably cooperated with arespectively-corresponding valve (one shown as 1460) that fits into ajudiciously dimensioned valve port (shown as 1460A) configured in thelayer 1420. FIG. 14A schematically illustrates a top view of thestructural layer 1420 of the embodiment 1400, while FIG. 14C outlinesthe top view of the fluidic manifold layer 1440.

Each of the embodiments 1200, 1300, and 1400 is illustrated to containmultiple individual channels sharing the main outlet (optionally fluidlyconnected with a disposable external storage volume; not shown). It isunderstood, however, that a related implementation (not shown) can bestructured to ensure that individual outlet portions of at least some ofthe constituent channels of the microfluidic network chip are directingthe flow of corresponding fluidic sample(s) directly to the externalstorage volume and not to the shared main outlet channel portion.

The operation of any of the embodiments of FIGS. 12, 13A, 14A accordingto the general principle outlined in connection with FIG. 11 produced anempirical result already summarized in FIG. 8. Data presented in FIG. 8evidence the reduction and/or elimination of spatial drift of thefluidic sample(s) by temporarily and reversibly bringing the fluidicsample(s) to a standstill (that is, stopping and/or restraining and/orspatially stabilizing the sample(s)) with no air present within theboundaries of the sample(s) for the duration of a photometricmeasurement as a result of causing the sample(s) in fluidic channel(s)to interact with pressure forces simultaneously applied to opposite endsof such sample(s) by respectively-corresponding fluidic valve(s) andauxiliary fluid(s) such as gas. As a result of the implemented solution,the persisting problems caused to the photometric measurement inembodiments of related art—that is, the presence of large modulations ofmeasured intensity values below and/or above the floor of themeasurement, leading to uncertainties of how and where to averageacquired data to obtain physically-correct results—was solved. Asevidenced by FIG. 8, as a result of implementing an embodiment of theinvention not only the abrupt (see curve portion 820A) but also acontinual-in-time drift (see curve portion 840) of the fluidic sample issubstantially eliminated, leading to increased accuracy and precision ofthe now-reliable analytical measurement(s). A skilled artisan willreadily appreciate that, when a specific embodiment of the inventionincorporates a dedicated valve at the output portion of each of thechannels, such incorporation additionally solves the problem ofcross-contamination between the contents of first and second constituentchannels of the network of channels regardless of which level of fluidicresistance each of such channels possesses in comparison with otherchannel(s).

The understanding of any of the embodiments of FIGS. 12, 13A, 14A (orrelated embodiments) will be further enhanced upon considering theprinciple according to which the fluidic sample of interest isentered/delivered to the microfluidic chip such as chip 1200, 1300,1400, for example. To this end, the following discussion, presented inreference to FIGS. 15A, 15B, 15C, and 15D and in further reference toFIGS. 9, 10A, 10B, provides additional insights into the system of theinvention and its principle of operation. FIGS. 15A, 15B, 15C, and 15Dillustrate a portion of an embodiment of the system located upstreamwith respect to a cuvette of an individual, constituent channel of themicrofluidic chip (such as the cuvette 910C, for example) and timedoperation of this portion, leading to the filling of the individualcuvette with a fluidic sample of interest and locking of such sample inplace to eliminate the spatial drift of the sample during thephotometric measurement.

In particular, FIGS. 15A, 15B, 15C, and 15D schematically illustrate a“store and forward” well or individual volume 1510, which is locatedupstream with respect to and is fluidly connected with the inlet portion910A of the individual channel of the microfluidic chip. Also shown is amulti-way fluidic valve 1520 preceding the will 1510, through which afluidic connection is operably established from a pump (as shown by anarrow; for example, pneumatic pump or stored pressurized gas cylinder,configured to deliver, in operation of the system, an auxiliary fluidtowards the well 1510 and then towards the cuvette 910C to form apressure front 1010) to the well 1510 and further through the individualchannel to the valve 960. The well 1510 is dimensioned to hold/enclosetherein a sample volume required (as shown in FIG. 10B) for properoperation of an embodiment of the invention, and has its inlet oraperture 1510A covered by a flap portion 1510B.

In one implementation, the flap portion is configured such as tosubstantially impenetrably seal the aperture of the well 1510, frominside the well, when the flap 1510B is in a rest position, see FIG.15A. In other words, the flap 1510B is configured to substantiallyprevent a fluid from ingressing/egressing the volume of the well 1510through the aperture 1510A connecting this volume with the ambientmedium. (As will be understood from the further description, thisembodiment of the well is preferably used with the 2-way version of thevalve 1520, as discussed below). In a related embodiment, the flap 1510Bis configured such as to provide for residual fluid seepage or leakingbetween the flap and the rims of the aperture 1510A in the directionfrom outside of the well 1510 to inside of the well 1510. (Thisembodiment is preferably used when the valve 1520 is configured as a3-way valve, as shown below.) In either of the cases, however, thecooperation between the flap 1510B and the aperture 1510A ismechanically structured to sufficiently restrict the fluid flow out ofthe well 1510 and to have the pressure inside the well build up. Theflap 1510B and the aperture 1510A are mechanically cooperated to ensurethat—when the flap is in the rest position, covering the opening of theaperture to close the aperture—the residual flow of fluid through theaperture into the well is greater that the residual flow of fluidthrough the aperture out of the well. Generally, the flap 1510B can bestructured as a elastomeric element (deformable within the limits ofelastic deformation of the material of the flap, which may be, forexample, a silicone rubber) or a rigid-material based spring-loaded flap(optionally complemented with a O-ring gasket disposed between the rigidflap and the rim of the aperture to improve sealing between the flap andthe well 1510A; not shown). In the latter case, the spring loading theflap is configured to operate within the limits of its elasticdeformation.

In operation, a sample of interest 1020 is delivered to the well 1510through the aperture 1510A with a use of a pipette 1524 (of a roboticpipette system; not shown) that is judiciously inserted into the well1510 through the aperture by forming a contact between the tip of thepipette 1524 to apply pressure to and appropriately deflect the flap1520B, as shown in FIG. 15B. Before the insertion, the multiway valve1520 is kept closed to ensure that no fluidic pressure is delivered fromthe pump to the internal volume of the well 1510. Once the tip of thepipette 1524 is in the internal volume of the well 1510, a prescribedamount of the sample 1020 is added to the well 1510 while, at the sametime, the multi-way valve 1520 is kept closed to block the propagationof the auxiliary fluid 1528 through the valve 1520 and towards thevolume of the well 1510. Following the disbursement of the sample 1020into the well 1510, the pipette 1524 is removed from the well 1510 toallow the flap 1510B to assume its rest position and substantially sealthe aperture 1510A.

Now, in reference to FIG. 15C, the multi-way valve 1520 is opened toallow the pressurized auxiliary fluid 1528 (such as gas, for example)pass the valve 1524, fill the well 1510 while pushing the sample 1020towards the inlet portion 910A of the individual microfluidic channeland further drive the sample 1020 into the cuvette 910C. The numeral1530 indicates an interface between the fluid 1528 and the sample 1020,formed in an intermediate portion of the channel; leading to the cuvette910C. The propagation of the fluids 1020, 1528 downstream (i.e., in adirection from the well 1510 towards the cuvette 910C) coincides withand is balanced by another fluid 1534 present in the system downstreamfrom the cuvette and passing through the open valve 960.

In reference to FIG. 15D, once the sample 1020 has filled the cuvette910C and has been delivered past the valve 960 while pushing the fluid1534 downstream (as was already discussed in reference to FIG. 10B), thedownstream valve 960 is closed to isolate the sample 1020 in the system.The “pinching” of the fluid path by the valve 960 prevents the sample1020 from moving with respect to the cuvette 910C. At the same time, theupstream multi-way valve 1520 is partially closed/partially open, asshown in FIG. 15D, and due to the lack of continued entrance of thefluid 1528 into the system the pressure upstream of the sample dropssubstantially to a level of atmospheric pressure. Alternatively, asdiscussed above, the multi-way valve may continue to operate tocontinually apply the pressure 1010 to the sample 1020 to ensure theimmobilization of the sample with respect to the cuvette and tostore/preserve a level of pressure required for the removal of thesample from the cuvette upon the completion of the measurement. In thissituation, the following after the photometric measurement step ofremoving the sample of the cuvette 910C requires a reduced amount offluid 1528. In yet another related embodiment, the flap 1510B can beconfigured to not completely seal the aperture 1510A such that, afterthe valve 960 is closed, the flap seeps or oozes a bit to allow thefluidic pressure applied to the sample 1020 from upstream the cuvette tobe substantially at an atmospheric pressure level. Generally, andaccording to the idea of the invention, a) in a situation whenatmospheric pressure is sufficient to “lock the fluid sample in thecuvette”, the pressure created upstream to move the fluid in the cuvettecan be relieved, while b) when atmospheric pressure may be insufficientto “lock the fluid sample in the cuvette” or should the stored gaspressure be used to remove the fluid sample from the cuvette, thepressure created upstream with respect to the cuvette is maintained.

Additional illustrations to the implementation of concepts of theinvention are provided by schematic diagrams of FIGS. 15E, 15F, 15G, and15H, in which the inlet/outlet portions (such as those shown as 1560,1564) of individual microfluidic channels are shown in solid lines, thecuvette portions (such as 1568) are shown with circles, the valves (suchas 1572) are indicated with “bow-tie” indicia, and the main outletchannel portions (if present, such as 1576, delivering measured fluidsamples from individual cuvettes to the common waste storage) are shownwith wide stripe-shaped rectangles. Fluidic resistance of an i-thportion of a channel is indicated with Ri. In the schematics of FIGS.15E and 15F, portions of channels characterized by R₁ are designed toact as capillary valve(s) for the cuvette(s), while the differencebetween R₂ and R₃ values is intended to be and is chosen to be, inpractice, substantial enough to ensure that backflow into any of thechannel portions with R₁ is eliminated (path of least resistance). Inthe embodiments structured according to the schematics of FIGS. 15G,15H, channel portions with R1 reduce flow rate (due to, for example,increased pressure drop that is a function of channel diameter andlength) at any given input pressure, in addition to minimizing a volumeof the sample used for the measurement. This improves the ability of thesystem to control the fluid through the valve with respect to utilizingthe act of closing the valve in order to minimize the used fluid samplevolume.

The operation of the embodiment(s) of the invention has been validatedby photometrically measuring fluidic (blood) sample(s) contained inidentified cuvette portion(s) of the channels and isolated fluidicallyfrom the rest of the system to simultaneously andindependently-for-each-cuvette control the time-variable processes(assay chemistry) to determine the concentration of hemoglobin, glucose,and alkaline phosphatase (ALP). (Notably, the ability to control andgovern and effectuate transfer of the fluid(s) through one channel orportion of the channel of the microfluidic chip independently from theprocess of transfer through another channel allows the user of theembodiment of the invention to carry out different measurements indifferent cuvettes at the same time or according to time-overlappingschedules—and regardless of the nature of the measurements. Differenttypes of chemical reactions may take different times and may requiredifferent starting and/or processing conditions such as temperature, forinstance. Non-limiting examples of such reactions are the rate reactionand enzymatic reaction. In case of inability of an embodiment of theinvention to independently control the timings ofdelivery/holding/flushing of the samples through the individualchannels, one of these two types of measurements would not be completeby the time another has already reached its termination point.)

The same samples had been previously measured with the use ofconventional clinical laboratory diagnostic equipment specifically,Abbott Architect c4000). The results of comparison between these twomeasurements are presented in FIGS. 16A, 16B, and 16C, where the resultsof a measurement carried out with conventional equipment are referred tothe x-axes of the plots, while the results of the measurement with anembodiment of the present invention are referred to the y-axes of theplots. The exceptional performance of the system of the invention isevidenced not only by the substantial linearity of the comparisonregardless of the value of concentration (mg/dL), but also by the factthat such linear dependencies are represented by straight lines inclined(with respect to x- or y-axes) by about 45 degrees. A person of skill inthe art will immediately recognize that the coefficient of variationbetween the two instruments (or, the results of measurements conductedwith the use of two methodologies) is substantially equivalent and iswell within the values of experimental errors. The results achieved withthe use of the methods of the present invention are statisticallyequivalent to those obtained with conventional equipment, as validatedby a third party.

To effect the operation of an embodiment of the above-described IPMsystem (including the design of a multiplexed microfluidic chipaccording to the methodology described above) and performance of thesteps required to acquire and process the photometric data representingresults of the measurements of the fluid sample(s) passing through anindividual cuvette of the IPM system may require the operation of aprocessor controlled by application-specific instructions stored in atangible memory element. Those skilled in the art should readilyappreciate that required algorithmical functions, operations, anddecisions may be implemented as computer program instructions, software,hardware, firmware or combinations thereof. Those skilled in the artshould also readily appreciate that instructions or programs definingthe functions and elements of the present invention may be delivered toa processor in many forms, including, but not limited to, informationpermanently stored on non-writable storage media (e.g. read-only memorydevices within a computer, such as ROM, or devices readable by acomputer I/O attachment, such as CD-ROM or DVD disks), informationalterably stored on writable storage media (e.g. floppy disks, removableflash memory and hard drives) or information conveyed to a computerthrough communication media, including wired or wireless computernetworks. In addition, while the invention may be embodied in software,the functions necessary to implement the invention may optionally oralternatively be embodied in part or in whole using firmware and/orhardware components, such as combinatorial logic, Application SpecificIntegrated Circuits (ASICs), Field-Programmable Gate Arrays (FPGAs) orother hardware or some combination of hardware, software and/or firmwarecomponents.

Within this specification, embodiments have been described in a way thatenables a clear and concise specification to be written, but it isintended and will be appreciated that embodiments may be variouslycombined or separated without parting from the scope of the invention.In particular, it will be appreciated that each of the featuresdescribed herein is applicable to most if not all aspects of theinvention.

The invention as recited in claims appended to this disclosure isintended to be assessed in light of the disclosure as a whole, includingfeatures disclosed in prior art to which reference is made.

For the purposes of this disclosure and the appended claims, the use ofthe terms “substantially”, “approximately”, “about” and similar terms inreference to a descriptor of a value, element, property orcharacteristic at hand is intended to emphasize that the value, element,property, or characteristic referred to, while not necessarily beingexactly as stated, would nevertheless be considered, for practicalpurposes, as stated by a person of skill in the art. These terms, asapplied to a specified characteristic or quality descriptor means“mostly”, “mainly”, “considerably”, “by and large”, “essentially”, “togreat or significant extent”, “largely but not necessarily wholly thesame” such as to reasonably denote language of approximation anddescribe the specified characteristic or descriptor so that its scopewould be understood by a person of ordinary skill in the art. In onespecific case, the terms “approximately”, “substantially”, and “about”,when used in reference to a numerical value, represent a range of plusor minus 20% with respect to the specified value, more preferably plusor minus 10%, even more preferably plus or minus 5%, most preferablyplus or minus 2% with respect to the specified value. As a non-limitingexample, two values being “substantially equal” to one another impliesthat the difference between the two values may be within the range of+/−20% of the value itself, preferably within the +/−10% range of thevalue itself, more preferably within the range of +/−5% of the valueitself, and even more preferably within the range of +/−2% or less ofthe value itself. The term substantially equivalent is used in the samefashion.

The use of these terms in describing a chosen characteristic or conceptneither implies nor provides any basis for indefiniteness and for addinga numerical limitation to the specified characteristic or descriptor. Asunderstood by a skilled artisan, the practical deviation of the exactvalue or characteristic of such value, element, or property from thatstated falls and may vary within a numerical range defined by anexperimental measurement error that is typical when using a measurementmethod accepted in the art for such purposes.

The disclosed embodiments of the invention discuss specific examples ofisolation of multiple different sample chemistries within a singleconnected fluidic network, of design rules for optimizing physicalcharacteristics of the measurement cuvettes and fluidic network, ofdesign rules for cuvettes to be repeatedly used in a fluidic network(including sample loading without air bubbles and sample flushingwithout carryover contamination), and of performing simultaneousphotometric measurements of (optionally multiple) samples at (optionallymultiple) wavelengths in a consolidated package. Modifications to, andvariations of, the illustrated embodiments may be made without departingfrom the inventive concepts disclosed herein. Furthermore, disclosedaspects, or portions of these aspects, may be combined in ways notlisted above. Accordingly, the invention should not be viewed as beinglimited to the disclosed embodiment(s). In addition, the terminologyused herein is with the purpose of describing particular embodimentsonly, and is not intended to limit the scope of the present invention.

What is claimed is:
 1. A microfluidic device comprising: first andsecond substrates integrated with one another along surfaces thereof toform a stack of substrates; a first microfluidic channel including firstinlet portion, first cuvette portion, and first outlet portion, whereinat least one of said first inlet and first outlet portions traversesboth of said first and second substrates; a first fluidic valve in fluidcommunication fluidly connected to the outlet portion; a fluidic welldisposed upstream with respect to the first cuvette portion in fluidcommunication with the first inlet portion, said fluidic well having aninternal volume and an aperture connecting said internal volume with anambient medium surrounding the fluidic well, said fluidic well equippedwith a flap element dimensioned to reversibly close the aperture frominside the fluidic well when in a rest position, and to reversibly opensaid aperture in response to a force applied to the flap element fromthe ambient medium inwardly to the internal volume; and at least one ofthe following: a second fluidic valve that is disposed upstream withrespect to said fluidic well and configured to deliver an auxiliaryfluid into the fluidic well along a channel connecting the secondfluidic valve with the fluidic well; a second microfluidic channelincluding a second inlet portion, a second cuvette portion, and a secondoutlet portion, wherein at least one of said second inlet and outletportions extends across both the first and second substrates; and a mainoutlet channel fluidly connected to both the first and second inletportions to receive a fluid sample from either of said first and secondcuvette portions.
 2. The microfluidic device according to claim 1,configured to ensure that transfer of fluid through the firstmicrofluidic channel and transfer of fluid through the secondmicrofluidic channel are independent from one another.
 3. Themicrofluidic device according to claim 1, further comprising a storagevolume in fluid communication with the main outlet portion to receivesaid fluid sample from either of the first and second cuvette portionsthrough the main outlet channel.
 4. The microfluidic device according toclaim 1, wherein said main outlet channel penetrates both the first andsecond substrates.
 5. The microfluidic device according to claim 1,wherein said first cuvette portion is dimensioned to prevent formationof an air-bubble therein when a first fluid is being delivered from thefirst inlet portion to the first cuvette portion.
 6. The microfluidicdevice according to claim 1, further comprising at least one of thefollowing: a) a third substrate in said stack, wherein at least two ofthe first, second, and third substrates are integrated with one anothervia a surface sealing layer, the surface sealing layer configured tofluidly seal a junction, wherein said junction is formed by the at leastone of the first inlet and outlet portions traversing said at least twosubstrates; and b) a light source configured to deliver a beam of lightto said first cuvette; and an optical detector disposed in opticalcommunication with said first cuvette such as to receive at least aportion of said beam that has traversed the first cuvette.
 7. Themicrofluidic device according to claim 6, wherein the light source isconfigured to deliver additional light to said second cuvette; andwherein the optical detector is disposed in optical communication withsaid second cuvette such as to receive at least a portion of saidadditional light that has traversed the second cuvette.
 8. Themicrofluidic device according to claim 1, wherein at least one of saidsecond inlet and second outlet portions traverses both of said first andsecond substrates.
 9. A microfluidic device comprising: first and secondsubstrates integrated with one another along surfaces thereof to form astack of substrates; a first microfluidic channel including first inletportion, first cuvette portion, and first outlet portion, wherein atleast one of said first inlet and first outlet portions traverses bothof said first and second substrates; a first fluidic valve in fluidcommunication fluidly connected to the outlet portion; a fluidic welldisposed upstream with respect to the first cuvette portion in fluidcommunication with the first inlet portion, said fluidic well having aninternal volume and an aperture connecting said internal volume with anambient medium surrounding the fluidic well, said fluidic well equippedwith a flap element dimensioned to reversibly close the aperture frominside the fluidic well when in a rest position, and to reversibly opensaid aperture in response to a force applied to the flap element fromthe ambient medium inwardly to the internal volume; wherein said firstcuvette portion is dimensioned to prevent formation of an air-bubbletherein when a first fluid sample is being delivered from the firstinlet portion to the first cuvette portion.
 10. The microfluidic deviceof claim 9, comprising at least one of the following: a second fluidicvalve that is disposed upstream with respect to said fluidic well andconfigured to deliver an auxiliary fluid into the fluidic well along achannel connecting the second fluidic valve with the fluidic well; asecond microfluidic channel including a second inlet portion, a secondcuvette portion, and a second outlet portion, wherein at least one ofsaid second inlet and outlet portions extends across both the first andsecond substrates; and a main outlet channel fluidly connected to boththe first and second inlet portions to receive fluid from either of saidfirst and second cuvette portions.
 11. The microfluidic device accordingto claim 10, configured to ensure that transfer of liquid through thefirst microfluidic channel and transfer liquid through the secondmicrofluidic channel are independent from one another.
 12. Themicrofluidic device according to claim 10, further comprising a storagevolume in fluid communication with the main outlet portion to receivesaid fluid from either of the first and second cuvette portions throughthe main outlet channel.
 13. The microfluidic device according to claim10, further comprising at least one of the following: a) a thirdsubstrate in said stack, wherein at least two of the first, second, andthird substrates are integrated with one another via a surface sealinglayer, the surface sealing layer configured to fluidly seal a junction,wherein said junction is formed by the at least one of the first inletand outlet portions traversing said at least two substrates; and b) alight source configured to deliver a beam of light to said firstcuvette; and an optical detector disposed in optical communication withsaid first cuvette such as to receive at least a portion of said beamthat has traversed the first cuvette.